Polyethylene glycol microparticles are highly biocompatible and have numerous advantages over conventional polymeric (polystyrene, latex, etc.) beads. For instance, PEG microparticles have low autofluorescence, are porous, have low non-specific binding, and can have different functionalities. In particular, PEG with reactive acrylate groups can be utilized to form hydrogel particles using ultraviolet (UV) exposure. PEG microparticles thus have desirable attributes for biological applications.
PEG particles are readily formed by UV curing of acrylate functionalized PEG. PEG-monoacrylate (PEGMA) and PEG-diacrylate (PEGDA) are common forms of UV curable PEG. This involves the use of a photoinitiator with an acrylate functionalized PEG mixture. Exposure to UV leads to the initiation of the reaction and thus the formation of the particles.
A common challenge to all PEG microparticles is functionalization. PEGDA or PEGMA does not have amine, sulfhydryl, or other chemical groups that can be readily utilized for attachment of biomolecules. Proteins and other biomolecules without functional groups can be directly polymerized into the PEG without functional groups. The downside of this approach is that the biomolecules may leach out from the PEG matrix since they are not covalently attached. Another downside, particularly for proteins, is that mixing them with PEG leads to protein precipitation. PEG is a common molecule utilized to precipitate proteins for a variety of applications.
One approach to incorporating biomolecules into the PEG matrix includes reacting the protein or biomolecule with a heterobifunctional PEG molecule, such as ACRYL-PEG-SCM, prior to mixing the protein in with the PEG mixture, where SCM stands for succinimidyl carboxymethyl. The SCM group reacts with the amine groups on the proteins, attaching the PEG-acrylate molecule to it and making it more soluble in a PEG mixture. Conventionally, proteins are precipitated in PEG, but this approach allows mixture of the protein with PEG. This approach, however, utilizes significant amounts of protein, as much as 25 μg/μL, which is highly impractical. Furthermore, different types of protein may precipitate in the PEG mixture.
Amine-based chemistries are challenging because they are highly labile. Groups with SCM have half-lives of 0.75 minutes and those with succinimidyl valerate have 33.6 minutes. While these groups can be introduced into the PEG mixture, they will be hydrolyzed by the time the PEG microparticle synthesis is complete and this therefore is not the best solution. Rapid hydrolysis is therefore a hurdle to these chemistries.
At least one group has attempted to add carboxyl groups to the PEG matrix by introducing acrylic acid into the PEG-based mixture. The polymerization of acrylic acid into the particles results in carboxyl groups that can later be functionalized. Conventionally, carboxyl groups can be coupled to amine groups on biomolecules using EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-Hydroxysuccinimide). This chemistry results in amide bond formation between the protein and the hydrogel surface. While this is a common approach for the addition of biomolecules to carboxyl groups, the chemistries are highly sensitive to moisture, requiring them to be stored in dry environments, and even with desiccants the reagents can degrade quickly.
Another impediment to the additional of functional groups to the hydrogels is the commercial availability of heterobifunctional PEG molecules. The commercial stock of various types of heterobifunctional PEG molecules changes with time and thus limits the type of functionalized PEG microparticles that can be fabricated.
For the foregoing reasons, there are significant challenges that need to be overcome in order to create a stable coupling chemistry for PEG-based microparticles.